DESCRIPTION: The objectives of the proposed work are to contribute to an understanding of the molecular basis for the function of the high valent hemes in unstable peroxidase intermediates using resonance Raman spectroscopy. Structural aspects will be addressed using rapid acquisition resonance Raman techniques which involve the rapid mixing of enzyme, oxidant, and substrate. The products are probed by laser excitation before they can decay into more stable forms. The proposed work will focus on chloroperoxidase from Caladariomyces fumago and yeast cytochrome c peroxidase for which crystallographic data are available. The aim is to develop a picture of the interaction of the amino acid residues lining the heme pocket with the activated oxidized hemes in the intermediate states by monitoring variations in specific resonance Raman frequencies as a function of pH and other variables. In cytochrome c peroxidase such effects will be delineated for the oxidized ferryl intermediates using site specific mutations and isotopic substitution. Chloroperoxidase possesses structural and functional similarities to the cytochrome P-450 enzymes. The P-450 enzymes catalyze the hydroxylation of apolar xenobiotics but are also believed to be involved in the activation of carcinogens. A long term goal of the proposed work is to contribute to the understanding of the molecular aspects of the cytochrome P-450 mechanism for which chloroperoxidase intermediates are considered as important models. The chlorinating activity of chloroperoxidase parallels the physiological defense mechanisms of mammalian peroxidases, such as myeloperoxidase and eosinophil peroxidase, which are key components of the immune response. These enzymes can also cause damage to host tissue. Though the peroxidation of chloride ion is central to the activity of these enzymes, the molecular nature of the active oxidizing species is not yet understood.